This invention relates to methods for producing highly purified mixtures of natural Type I interferon from white blood cells, with a protein purity in excess of 95% as determined by standard analytical methods. The invention also relates to highly-purified mixtures of Type I interferon which resemble natural Type I interferon in that the highly purified mixtures of natural Type I interferons includes at least 9 subtypes, i.e., alpha-1, alpha-2, alpha-5, alpha-7, alpha-8, alpha-10, alpha-14, alpha-21 and omega. These subtypes may be present as varying proportions of the following subspecies: alpha-1a, alpha-1new, alpha-2a, alpha-2b, alpha-2c, alpha-5, alpha-5LG, alpha-7, alpha-8a, alpha-8c, alpha-10a, alpha-14a, alpha14b, alpha 14-c, alpha-14LG, alpha-21a, alpha-21b, alpha-21c, omega and omega LG.
The interferons are a family of proinflammatory cytokines important in mediating nonspecific host defense. While of critical importance in initiating anti-viral immunity, the family also acts as a potent initiator of cell growth and differentiation. Type I interferon is a designation for a family of related interferons that can include multiple subtypes of alpha, beta and omega interferons, and in some species the related trophoblast tau interferon. The proteins are structurally similar, share common receptors, have common biological activities and may share a common genetic locus.
Type I interferon is believed to have three major functions. First, it inhibits viral replication by activating cellular genes that inhibit protein synthesis, thus contributing to the suppression of viral replication. Second, Type I interferon downregulates the proliferation of specific cell types, a characteristic applied to the treatment of certain cancers. Finally, Type I interferon has an immunomodulatory effect, which can influence the nature of the immune response (i.e. cellular or humoral) while activating innate components such as NK cells or monocytes.
The plurality of effector functions of Type I interferon creates a variety of potential pharmacological applications. While recognized for its antiviral capability, Type I interferon is also effective in the treatment of some bacterial and eukaryotic pathogens. In addition, the immunomodulatory properties of the subtypes in Type I interferon have proven useful in the treatment of some cancers and autoimmune disorders. The literature describing the uses of interferon preparations is vast and includes the use of Type I interferon in the treatment of cancers, including leukemias (U.S. Pat. No. 5,830,455), basal cell carcinomas (U.S. Pat. No. 5,028,422), squamous cell carcinomas (U.S. Pat. No. 5,256,410), breast cancer (U.S. Pat. No. 5,024,833), gastrointestinal malignancies (U.S. Pat. No. 5,444,064; 5,814,640), actinic keratoses (U.S. Pat. No. 5,002,764), as well as macular degeneration (U.S. Pat. No. 5,632,984), autoimmune disorders (U.S. Pat. No. 5,830,456), diabetes (WO0980643 1A2), bacterial infections (U.S. Pat. No 5,817,307), and viral infections (U.S. Pat. No. 5,830,456), including genital warts (U.S. Pat. No. 4,959,210), hepatitis B (WO09823285A1), and herpes zoster and psoriasis (U.S. Pat. No. 4,957,734). While the pharmaceutical applications of this family of cytokines is only beginning to be understood, the problems related to obtaining an inexpensive and highly purified preparation containing a comprehensive spectrum of Type I interferon have limited the therapeutic potential of Type I interferon.
A number of different techniques have been utilized to produce quantities of interferons. The successful cloning and sequencing of genes encoding specific members of the family have allowed for the recombinant production of individual Type I interferon subtypes. While it is possible to produce individual recombinant Type I subtypes, these individual recombinant products are limited because (1) their structures may vary from the natural state, and (2) their individual activities may lack the therapeutic potential of all subtypes collectively. Further, individual interferon subtypes may cause negative host reactions, including fever, nausea, tissue necrosis and psychopharmacological effects. These side effects have in some cases limited the efficacy of interferon treatment.
Natural interferon production heretofore has traditionally involved ammonium chloride treatment of buffy coats to lyse red blood cells and to isolate leukocytes, followed by viral stimulation of the isolated leukocytes with subsequent large scale harvesting of culture medium. The interferons are then isolated by various precipitation, adsorption, or immuno-affinity techniques.
Despite the use of a variety of purification techniques, the quality, quantity and subtype diversity of the interferons obtained using these methods has remained unsatisfactory. These techniques have generally required tremendous quantities of culture media with processing resulting in low yields of product having limited subtype distribution. It is presently believed that the methods described heretofore are unable to achieve easily and economically a sufficiently high recovery rate with a high degree of purity, full spectrum of subtypes and full functional activity of natural interferon subtypes. Even immuno-based purification techniques have not produced a full spectrum of individual Type I interferon subtypes because the various subtypes differ in their antigenicity.
It is currently thought that one important reason for the low yield of Type I interferon from the prior art techniques is due to ineffective methods of leukocyte collection, transport, separation, culture and stimulation to secrete interferons. For example, transport mechanisms for whole blood or buffy coats are not optimal for retaining active leukocyte cells, being subject to a wide range of temperatures, high osmolarity, low oxygenation levels and variable transport times. Also, it is presently believed that lysis of red blood cells with ammonium chloride may greatly reduce the yield of leukocytes, many of which may also be lysed. The remaining leukocytes are then osmotically shocked which is thought to render the captured leukocytes less effective in their protein synthesis, further reducing the yield of product. Further, the prior art utilized hitherto usually employs serum in the culture of leukocytes, which is believed to significantly contribute to the resulting contamination of the secreted protein product. Moreover, it is thought that the use of viral preparations to induce interferon production also adds a significant source of contaminating material. To date, the prior art methods available up to now have not addressed these problems.
Turning now to representative interferon disclosures, U.S. Pat. No. 5,503,828 describes an alpha-interferon composition characterized by having at least 50% of alleles of xcex12 and xcex18, and one or more additional alpha interferon species selected from the group consisting of xcex14, xcex17, xcex110, xcex116, xcex117, and xcex121. While U.S. Pat. No. 4,503,035 teaches a preparation of certain interferon species, the preparation does not include, for example, alpha-1, alpha-5, alpha-14 and omega subtypes. Thus, a natural mixture of highly pure interferon having a full spectrum of subtypes is not taught by this U.S. Pat. No. 4,503,035.
U.S. Pat. No.5,762,923 teaches an aqueous interferon composition dissolved in water with a non-ionic detergent and benzyl alcohol in amounts sufficient to stabilize the interferon-alpha. The composition also contains an acidic buffer which provides a pH of 4.5 to 6.0, and may also contain an isotonizing agent. U.S. Pat. No. 4,847,079 teaches a pharmaceutical composition of interferon and thimerosal which is resistant to microorganism contamination and growth. U.S. Pat. No. 4,675,184 teaches an interferon with 15 to 60% by weight of a tri or higher polyhydric sugar alcohol and an organic acid buffer as stabilizers, and a conventional pharmaceutical carrier or diluent at pH about 3 to 6. Optionally, the composition may further contain an anionic surfactant and/or albumin as a stabilizer. U.S. Pat. No. 5,236,707 teaches the use of amine stabilizing agents, such as primary aliphatic amines, and anionic stabilizing agents, such as lithium organo sulfates, which according to this disclosure protect human interferons from degradation and provide enhanced storage stability. Similarly, U.S. Pat. No. 5,431,909 teaches the use of amine stabilizing agents, such as primary aliphatic amines, and anionic stabilizing agents, such as lithium organo sulfates, to protect human interferons from degradation and provide enhanced storage stability.
U.S. Pat. No. 4,780,413 relates to the production of interferon from lymphoblastoid cells by adding an inducer to lymphoblastoid cells. This interferon product appears to be marketed by Burroughs-Wellcome under the name WELLFROM(copyright), and is reported to contain 9 different subtypes and up to 22 molecular species. U.S. Pat. No. 4,172,071 relates to a process for the purification of interferon by absorption onto chromophore blue columns and elution with a low salt buffer. U.S. Pat. No. 4,289,689 combines affinity chromatography with high pressure liquid chromatography to purify interferons from fibroblasts. U.S. Pat. No. 4,465,622 describes a method of adsorbing interferon onto a carrier containing acrylonitrile polymer and eluting the adsorbed interferon with an appropriate buffer. U.S. Pat. No. 4,485,017 discloses a process wherein a partially purified preparation is passed through an antibody affinity column and a reversed-phase high performance liquid chromatographic column. Organic solvents used during the elution are extracted and the protein concentrated for subsequent use. U.S. Pat. No. 4,551,271 describes the purification of solutions of recombinant interferons by chromatography on metal chelate resins, including copper or nickel. U.S. Pat. No. 5,391,713 describes a process for purification of human leukocyte interferon which includes immunoaffinity chromatography, ion-exchange chromatography, and a series of precipitation and centrifugation steps.
While these disclosures provide different methods for obtaining an interferon preparation, they fail to provide a naturally-occurring Type I interferon product derived from white blood cells, which has a high degree of purity, a full spectrum of subtypes and fall functional activity of natural Type I interferon, and as a result, such interferon preparations available hitherto have limited yield and utility. Moreover, these processes are believed to be inefficient and quite expensive.
Consequently, there is a need for a highly-purified preparation of Type I interferon that resembles natural Type I interferon and which contains a full spectrum of interferon subtypes, particularly applicable to therapeutic uses. Moreover, there is a need for a process that can obtain high yields of a highly-purified preparation of Type I interferon in a simple yet efficient manner, so that the inefficiencies and inconveniences faced with interferon purification procedures available heretofore are overcome.
The present invention overcomes many of the shortcomings of the present state of the interferon art through the discovery of novel highly purified mixtures of Type I interferon derived from white blood cells, and novel methods of isolating and using same.
The multisubtype Type I interferons of the present invention are a highly purified blend of natural xcex1 and xcfx89 interferons obtained from leukocytes. The leukocytes may be derived from blood or a blood component, such as an apheresis product. No proteins, other than interferon proteins, can be quantifiably detecte multisubtype Type I interferons of the present invention using standard gel electropheresis techniques demonstrating that a purity equaling or exceeding 95% (w/w) is achieved. The multisubtype Type I interferons of the present invention are virtually free of contaminants, such as serum albumin, cytokines, and other low or high molecular weight proteins, in addition no nucleic acid (DNA/RNA) contamination has been found.
The molecular weights of the multisubtype Type I interferons of the present invention are generally between about 10,000 and about 30,000 Daltons, and more particularly between about 19,000 and 27,000 Daltons, as measured by SDS-Page. The multisubtype Type I interferons of the present invention have an activity of at least about 1xc3x97108 units, as measured by a standard anti-viral assay containing an international interferon standard, and apparent isoelectric points of between about 5.0 and about 8.5. Still further, the multisubtype Type I interferons of the present invention include both naturally glycosylated and naturally unglycosylated forms of interferon subtypes. The naturally glycosylated subtypes are believed to include alpha-2 species, alpha-14 species, alpha-21a and omega.
Uniquely, the multisubtype Type I interferons of the present invention resemble natural Type I interferon and in particular, natural human Type I interferon, in that they contain mixtures of multiple IFN-xcex1 and IFN-xcfx89 subtypes derived from white blood cells. More specifically, there are believed to be at least 9 different subtypes present that can give rise to at least 16, and possibly 20 or more molecular species. The 9 subtypes identified to date include alpha-1, alpha-2, alpha-5, alpha-7, alpha-8, alpha-10, alpha-14, alpha-21 and omega, whereas the 20 molecular species include alpha-1a, alpha-1new, alpha-2a, alpha-2b, alpha-2c, alpha-5, alpha-5LG, alpha-7, alpha-8a, alpha-8c, alpha-10a, alpha-14a, alpha14-b, alpha 14-c, alpha-14LG, alpha-21a, alpha-21b, alpha-21c, omega and omega LG. Because the multisubtype Type I interferons of the present invention are highly pure and contain a plurality or a significant number of different Type I interferon subtypes, it is believed that the multisubtype Type I interferons of the present invention very closely resemble the Type I interferon systems, which are naturally produced by and operating within animals and, in particular, the Type I interferon system naturally produced by and operating within humans, in both content of different subtypes and functional activity, especially when compared to the recombinant monocomponent interferon products available heretofore.
The present invention also contemplates novel procedures for obtaining the multisubtype Type I interferons. For example, the present invention is concerned with simple and efficient methods of obtaining a highly purified mixture of Type I interferon having a plurality of subtypes from leukocytes wherein the highly purified mixture of Type I interferon has a purity of at least about 95%, contains substantially only interferon proteins, contains at least 9 different subtypes, including alpha-1, alpha-2, alpha-5, alpha-7, alpha-8, alpha-10, alpha-14, alpha-21 and omega which gives rise to at least 16 and possibly 20 or more molecular species, and contains no more than about 35% by weight IFNxcex1-2 and IFNxcex1-8 subtypes. Generally speaking,such a method comprises: (a) culturing leukocytes; (b) stimulating the leukocytes to produce a crude interferon; (c) concentrating the crude interferon to remove low-molecular weight contaminants and liquid volume to produce a concentrated crude interferon; (d) removing a substantial amount of serum albumin and other contaminants from the concentrated crude interferon to produce a partially purified interferon mixture containing a plurality of subtypes; (e) removing substantially all remaining serum albumin and other contaminants from the partially purified interferon mixture to generate an interferon mixture having a purity of between about 50% and about 80%; and (f) purifying the about 50% to about 80% interferon mixture to produce a highly purified mixture of Type I interferon having a purity of at least about 95% and containing no more than about 35% by weight IFNxcex1-2 and IFNxcex1-8 subtypes.
Such a method may include the further step of isolating the leukocytes from blood or a blood component, such as an apheresis product.
Turning now to the novel procedures for obtaining the multisubtype Type I interferons of the present invention from leukocytes, they generally involve four segments: (1) leukocyte acquisition; (2) isolation of peripheral blood mononuclear cells (PBMC); (3) cell culture and interferon production in cell culture; and (4) purification of multisubtype Type I interferon.
Generally speaking, there are numerous advantages associated with these four segments. For instance, the acquisition of leukocyte segment results in an increase in the number of leukocytes due to better component manufacturing procedures. Additionally, the leukocytes are generally more hardy and have increased productivity due to the handling and isolation procedures contemplated by the present invention. Moreover, there is improved productivity of a xe2x80x9cunitxe2x80x9d of leukocytes due to (a) improved recovery from each donation, (b) additives added at the xe2x80x9cbuffy coatxe2x80x9d stage, and (c) temperature maintenance procedures. The additives into the buffy coat are believed to improve white blood cell health and separation. Notwithstanding, it should be appreciated that the procedure of the present invention is time sensitive from the nature of the donation to component manufacture to the PMBC isolation segment to IFN production, as developed further hereinafter. In addition, the procedures of the present invention require the use of gas permeable bags during storage of the harvested white blood cells and temperature maintenance (ambient, RT) during all steps leading from donation to culture.
As to the second segment, i.e., PMBC isolation, the PMBCs are generally healthier because the procedures of the present invention gently remove red blood cells from the white blood cells without lysis, the PMBCs are maintained at physiological ionic strength throughout the PBMC isolation process, the white blood cells are gently washed, and the PMBCs are gently isolated via use of a density separation media such as LSM or Ficoll Hypaque at manufacturing scale. Moreover, because plasma and plasma proteins are removed in accordance with the present invention, the use of non-immunoaffinity purification procedures can be used downstream to attain a purity of greater than about 95%. Finally, because the PMBCs are isolated, this limits proteolysis in cell culture.
During the third segment, i.e., cell culture and IFN production in cell culture, polymorphonuclear leukocytes (PMNs) are removed to minimize the adverse effects of protease. Further, use of purified Sendai Virus in cell culture and protein free medium are believed to enhance purification, limit the need for immunoaffinity, and remove any non-human proteins from the system. To enhance the benefit of this third segment, protease inhibitors should be used during cell culture, and suicide inhibitors of protease activity should also be used immediately after cell culture to help prevent product proteolysis. Moreover, pH, temperature, pO2, prevention of PMN breakdown, and cell health can be controlled during cell culture. Also, monocytes in the cell culture and presence of an inducing factor during the first two hours of cell culture after Sendai addition are generally needed.
With respect to the fourth segment, which concerns purification of the multisubtype Type I interferon, the protein burden of all steps leading to purification is reduced, so that non-immunoaffinity and non-RP-HPLC methods can be effectively employed for purification. This benefit reduces costs, limits structure degradation and enhances the recovery of Type I interferon subtypes. A reduction in protein burden using this fourth segment is accomplished by using protein free cell culture medium, purified Sendai, extensive washing of isolated white blood cells and removal of PMNs to prevent the release of their cellular contents into the purification stream. Moreover, the benefits of this fourth segment provide for the capture of a multisubtype Type I interferon which contains a mixture of multiple IFN-xcex1 and IFN-xcfx89 subtypes derived from white blood cells, which appears to resemble natural Type I interferon.
It should be appreciated that because the natural multisubtype Type I interferons of the present invention uniquely contain IFN-xcfx89 subtypes, it is believed that they should have anti-viral activity, in view of the fact that IFN-xcfx89 subtypes are well known for their strong anti-viral activity.
By way of illustrating and providing a more complete appreciation of the present invention and many of the attendant advantages thereof, the following detailed description and examples are given concerning the novel highly purified mixtures of Type I interferons derived from white blood cells in the present invention, and novel methods of isolating and using same.
In connection with the present invention, the following terms shall have the following meanings.
BCxe2x80x94Buffy coat.
BCAxe2x80x94Assay used for protein concentration determination.
Capture poolxe2x80x94concentrated interferon prepared from a culture medium by, for example, Sepharose Big Bead chromatography.
Crude interferonxe2x80x94Any sample of Type I interferon that is less than 35% pure, including the culture media into which interferon is secreted and concentrated culture medium.
CVxe2x80x94column volume.
ELISAxe2x80x94Enzyme linked Immunosorbent Assay. Commercial ELISA kits for detecting various interferons are used herein.
HEPESxe2x80x94N-2-Hydroxyethylpiperazine-Nxe2x80x2-2-ethanesulfonic acid.
Natural Mixture of Type I IFN or Interferonxe2x80x94Natural mixture of Type I INF or interferon, or multisubtype Type I interferons, or any other similar phrase, refers to any natural Type I interferon obtained from white blood cells, which comprises a blend of at least nine sub-types, i.e., xcex11, xcex12, xcex15, xcex17, xcex18, xcex110, xcex114, xcex121, and xcfx89, giving rise to at least 16, and possibly 20 or more, different molecular species, including alpha-1a, alpha-1new, alpha-2a, alpha-2b, alpha-2c, alpha-5, alpha-5LG, alpha-7, alpha-8a, alpha-8c, alpha-10a, alpha-14a, alpha14-b, alpha 14-c, alpha-14LG, alpha-21a, alpha-21b, alpha-21c, omega, omega LG and/or others.
LSM(trademark)xe2x80x94Lymphocyte Separation Medium. Contains ficol and hypaque.
MESxe2x80x942-(N-Morpholino)ethanesulphonic acid.
PBSxe2x80x94phosphate buffered saline.
PBMCxe2x80x94peripheral blood mononuclear cells.
PEFABLOC(trademark)xe2x80x94A commercially available serine protease inhibitor or its equivalent. Also, PEFABLOC SC(trademark).
PERCOLL(trademark)A commercially available solution of low osmolarity ( less than 25 mOsm/kg H2O) with variously sized silica particles. Any equivalent may be used
PF68xe2x80x94Pluronic acid F-68.
RB-1xe2x80x94refined buffy coat. Prepared by washing crude buffy coats with PBS (see example 2).
RP-HPLCxe2x80x94Reversed phase high performance liquid chromatography.
RPMIxe2x80x94Nutrient medium that supports leukocyte culture.
Semi-permeable bagxe2x80x94Also semi-permeable container. Any container with a large surface area to volume ratio that allows gas to pass through the container walls.
Serum-free culture mediumxe2x80x94any culture medium that can be used to support the growth of leukocytes and their production of interferon, but that does not contain any fetal calf or other type of serum. This includes at least RPMI, and MEM as well as others known to those of skill in the art.
Trisxe2x80x94N-Tris(hydroxymethyl)aminomethane.
VSVxe2x80x94vesicular stomatitis virus. Used in a bioassay to assess interferon anti-viral activity.
One aspect of the current invention concerns a highly purified mixture of Type I interferons containing at least 9 subtypes which gives rise to at least 16, and possibly up to 20 or more, molecular species. The 9 subtypes include IFN-xcex11, IFN-xcex12, IFN-xcex15, IFN-xcex17, IFN-xcex18, IFN-xcex110, IFN-xcex114, IFN-xcex121 and IFN-xcfx89. The molecular species include IFN-xcex11a, IFN-1new, IFN-xcex12a, IFN-xcex12b and/or IFN-xcex12c, IFN-xcex15, IFN-xcex15LG, IFN-xcex17, IFN-xcex18a, IFN-xcex18c, IFN-xcex110a, IFN-xcex114a and/or IFN-xcex114b and/or IFN-xcex114c, IFN-xcex114LG, IFN-21a and/or IFN-xcex121b, IFN-xcex121c, IFN-xcfx89 and IFN-xcfx89LG.
It should be appreciated by those versed in this art that the amounts of the subtypes and the individual molecular species in a highly purified natural mixture of Type I interferon obtained in accordance with this invention vary depending upon the method selected to isolate the natural mixture of Type I interferon from the leukocytes. Moreover, while the methods described herein will isolate highly purified natural mixtures of Type I interferon from white blood cells, any method of preparing a natural mixture of interferons with the characteristics described herein will suffice. The term xe2x80x9cnatural mixturexe2x80x9d refers to the fact that the interferons are xe2x80x9cnativexe2x80x9d or xe2x80x9cnaturalxe2x80x9d, e.g., not recombinant, and that they are purified as a mixture from white blood cells, rather than as individual subspecies which are then recombined.
The subtype listing provided above lists only those subtypes identified to date. Additional sequencing and peptide mapping studies of the highly purified natural mixtures of Type I interferon isolated in accordance with the present invention may determine that subtypes IFN-xcex17, IFN-xcex18, IFN-xcex110, IFN-xcex114 include the additional subtypes IFN-xcex17a, IFN-xcex17b, IFN-xcex17c, IFN-xcex18b, IFN-xcex110 b. Additionally, subtypes IFN-xcex14a, IFN-xcex14b, IFN-xcex116, IFN-xcex117a, IFN-xcex117b, IFN-xcex117c, and IFN-xcex117d may also be present as suggested by preliminary experiments.
Currently, it is believed that the major subtypes are about 25% IFN-xcex11 (a and new), about 15% IFN-xcex12 (a and b and/or c), about 5% IFN-xcex15 (a and LG), about 5% IFN-xcex17, about 10% IFN-xcex18 (a and c), about 10% IFN-xcex110a, about 10% IFN-xcex14 (a, b and/or c), about 10% IFN-xcex121 (a, b and/or c) and about 5% IFN-xcfx89.
The highly purified natural mixtures of Type I interferon are stabilized in a buffer with the addition of about 1 mg/ml HSA. Although most prior art interferon solutions employ acidic buffers in which to formulate the interferon, it has been found that a neutral pH works best under the conditions described. It should also be understood that the natural mixtures of Type I interferon of the present invention are suitable candidates for standard freeze-drying techniques.
For best results, the interferon is kept in silanized vials at 4xc2x0 C. and the vials may be sparged with N2 if desired. Any biocompatible buffer can be used to formulate the interferon and additional excipients and/or active ingredients may be added as necessary for the use and/or mode of application.
Because the highly-purified natural mixtures of Type I interferon of the present invention contain a full spectrum of interferon subtypes which are substantially free of contaminating proteins thereby closely resembling the natural interferons produced by humans from leukocytes, they are particularly applicable to therapeutic uses. For example, the present invention contemplates a method of treating interferon-responsive diseases by administering an effective amount of a highly purified natural mixture of Type I interferon (IFN) isolated from white blood cells in accordance with the present invention in a pharmaceutically acceptable carrier, said natural mixture of Type I IFN being at least about 95% pure before being combined with said pharmaceutically acceptable carrier, and said natural mixture of Type I IFN comprising at least 9 subtypes which gives rise to at least 16, and possibly up to 20 or more, molecular species. The 9 subtypes include IFN-xcex11, IFN-xcex12, IFN-xcex15, IFN-xcex17, IFN-xcex18, IFN-xcex110, IFN-xcex114, IFN-xcex121 and IFN-xcfx89. The molecular species include IFN-xcex11a, IFN-xcex11new, IFN-xcex12a, IFN-xcex12b and/or IFN-xcex12c, IFN-xcex15, IFN-xcex15LG, IFN-xcex17, IFN-xcex18a, IFN-xcex18c, IFN-xcex110a, IFN-xcex114a and/or IFN-xcex114b and/or IFN-xcex114c, IFN-xcex114LG, IFN-xcex121a and/or IFN-xcex121b, IFN-xcex121c, IFN-xcfx89 and IFN-xcfx89LG.
The methods of the present invention concern those diseases or indications that are interferon-responsive and include, for example, hepatitis infection, such as hepatitis A infection, hepatitis B infection, hepatitis C infection, HIV infection, herpes zoster virus infection; influenza infection, common cold infections, hemorrhagic fever infections, genital warts, bacterial infections, chlamydia infection, Behcet""s disease, Churg-Strauss syndrome, leukemia, T-cell leukemia, hairy cell leukemia, chronic myeloid leukemia, melanoma, myofibromatosis, T-cell lymphoma, basal cell carcinomas, squamous cell carcinomas, renal cell carcinoma, colorectal carcinoma, non-small cell lung cancer, cervical cancer, breast cancer, gastrointestinal malignancies, actinic keratoses, macular degeneration, autoimmune disorders, diabetes, psoriasis, multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis, systemic lupus erythematosus and the like.
Consistent with the present invention, the highly purified natural mixtures of Type I interferon (IFN), when intimately admixed in a pharmaceutically acceptable carrier, may be administered topically, orally, parenterally, sublingually, buccally, by nasal inhalation, rectally, vaginally, aurally, or ocularly.
Turning now to the general procedure for the preparation and culture of leukocytes according to the present invention, it begins with collecting, transporting and separating leukocytes from other blood cell fractions. Traditionally, leukocytes have been collected as whole blood and stored in impermeable plastic bags at about 4 C. to about 25 C. temperature until processed into plasma and red blood cells. The white blood cell layer (buffy coat) is generally a discarded side product of the process which can be collected and then treated with ammonium chloride to lyse the contaminating red blood cells. The remained leukocytes are then cultured in media containing a serum such as fetal calf serum and activated with a viral inducer to produce interferon.
In this invention, it has been discovered that it is possible to greatly improve the viability and activity of the white cells by changing this process to maximize and protect them during the collection, transport, purification and the subsequent culturing of leukocytes. Generally, the procedure is as follows:
Whole blood is collected at various collection centers and centrifuged to produce a buffy coat. Collection of the buffy coats is done with a peristaltic pump and a specially designed manifold that is much gentler than a vacuum pump. An initial wash with PBS serves to remove both platelets and some serum contaminants as well. After centrifugation, the supernatant is removed with a peristaltic pump and specially designed aspirators. Then the cells are gradually brought to isotonic osmolarity with PBS washes.
The transport of blood or buffy coats, if necessary, should be done in such a way as to minimize temperature variations, time of transport and maximize the oxygenation of the cells. The use of semi-permeable plastic bags hung from the top of an insulating container so as to allow free flow of oxygen between and into the bags greatly increases the viability of the leukocytes. Further, the temperature should be maintained at 22xc2x0xc2x13xc2x0 C. This has been achieved with the use of water bags inside the insulating container. Of course, the time between collection and processing should be minimized.
Instead of lysing red blood cells with salt, the various cells in the buffy coat are separated by density centrifugation on an inert density separation matrix such as LSM(trademark) gradient with a second inert bead matrix (PERCOLL(trademark)) overlay with a slightly mixed LSM(trademark)/Percoll(trademark) interface. After centrifugation, the second cell layer, containing the PBMC, is recovered with a peristaltic pump, washed in PBS and then transferred into RPMI medium. The use of this procedure minimizes the number of granulocytes, thus reducing the protease contamination of later cultures. Throughout this procedure, it is important that the cells be maintained at a constant room temperature. Even a brief exposure to low temperatures can cause inadequate separation and lowered IFN production.
The PBMC cells are cultured in a serum-free medium, such as RPMI, in order to minimize contaminants throughout the later product purification procedures. For Type I interferon production, the cells are cultured with interferon primer at 37xc2x0 C. for about 2 hours. Sendai virus is added and the culture continued for another 2 hours. Then the temperature is smoothly and quickly dropped to 28xc2x0 C. and culture continued for about 14 hours. One half-hour before the end of culture, a protease inhibitor can be added, but this step is optional.
The Sendai virus that is used to stimulate interferon production is generally grown in the allantoic fluid of chicken eggs. This provides an additional source of contaminating proteins in the final interferon product. Therefore, in order to maximize product yield and purity, this additional source of contaminants is purified about 1000 fold by centrifugation of the Sendai virus on a potassium tartrate density gradient or by other methods known in the art.
After harvest of the interferon-containing culture media, the contaminating virus can be killed by incubation of the product at pH 2. Virus hemagglutination activity is eliminated in 30 minutes of low pH treatment. Further, interferon activity, as measured by anti-viral bioassay, is not negatively affected by up to 24 hours of acid treatment.
This interferon capture pool is the starting product for the purification procedures discussed, hereinafter.
Following the culture of the cells with the appropriate inducer, the supernatant is removed from the cells and initially concentrated 100 fold by cation exchange chromatography. Other means of concentration commonly practiced in the art are envisioned by the present invention, such as salt, precipitation, ultrafiltration, dialysis, gel filtration, affinity chromatography, electrofocusing or electrophoresis or a combination of two or more of the above techniques.
The concentrated interferon obtained from leukocyte culture is then purified in three steps as follows.
First, interferon is isolated from the concentrate by hydroxyapatite (HA) chromatography at a low pH in the range of about 4.9 to about 5.2, and more preferably at about pH 5.0. This removes about 98% of the major contaminant which is human serum albumin, and other contaminating proteins, and provides an approximately 10-fold purification and additional concentration. The partially purified interferon can be further purified by size exclusion chromatography (SEC) to remove the remaining human serum albumin and other minor contaminants. This is followed by application to an anion exchange (AX) chromatography column wherein the binding group is a quarternary amino group, or hydrophobic interaction chromatography (HIC) or both.
When (1) HA, (2) SEC and (3) AX or HIC or AX and HIC are performed in sequence, a natural interferon mixture is produced that is in excess of 95% (w/w) pure, and has the following characteristics: (a) it contains at least nine interferon subtypes giving rise to at least 16, and possibly 19 or more, molecular species, (b) a mixture of apparent molecular weights of between about 10,000 and about 30,000 Daltons and more particularly between about 19,000 and 27,000 Daltons, as measured by SDS-PAGE, (c) an activity of at least about or greater than 1xc3x97108 units, as measured by a standard anti-viral assay containing an international interferon standard, and (d) apparent isoeletric points of between about 5.0 and about 8.5. The natural mixture of Type I IFN or interferon is thought to include both naturally glycosylated and naturally unglcosylated forms of interferon subtypes. Most subtypes, however, are thought to be unglycosylated. The glycosylated subtypes are believed to include alpha -2 species, alpha-14 species, and omega species.
Further, the subtype characterization to date reveals that the natural mixture of Type I interferon comprises:
Further, when hydrophobic interaction chromatography (HIC) is included as the final purification step without the use of anion exchange, the natural mixture of Type I IFN or interferon may contain cytokine IL-6 in an amount of about 1/7000 to 1/500 of a clinically relevant therapeutic dose of IL-6.
Comparison with commercially available interferon preparations, such as WELLFERON(trademark) (Burroughs-Wellcome), ALFERON-n3(trademark) (Interferon Sciences, Inc.), ROFERON-2a(trademark) (Roche Laboratories) and INTRON-2b(trademark) (Schering-Plough) reveals that these commercial preparations, in contrast, completely lack subtype-omega (as assayed by ELISA (Bender Wein)). Moreover, because these interferon preparations do not contain a full spectrum of subtypes derived directly from white blood cells, it is believed that they cannot resemble the natural Type I intereferon produced by leukocytes within the body.
It should be understood that when anion exchange is selected as the final step in the procedure in accordance with the present invention (HA, SEC, AX), IL-6 is removed. In addition, the amounts of the subtypes omega, alpha-14 and alpha-21 in the natural mixture of Type I IFN or interferon are substantially reduced. If the subtype omega is removed, the isoeletric point range changes from between about 5.0 to about 8.5 to between about 4.0 to about 6.0. Of course, the relative ratios of the subtypes in the natural mixture of Type I IFN or interferon produced by this method are adjusted appropriately by the removal of significant amounts of subtypes omega, alpha-14 and alpha-21.
It should also be understood that when anion exchange and HIC are selected as the final step in the procedure in accordance with the present invention (HA, SEC, AX and HIC), only IL-6 is removed. Again, the relative ratios of the subtypes in the natural mixture of Type I IFN or interferon produced by this method, i.e., HA, SEC, AX and HIC, are adjusted appropriately by the removal of IL-6.
In order that the invention and its advantages may be more completely understood, the following examples are provided as a means of illustration but are in no way considered as a limitation on the scope of the present invention.